Bergman, Yehudit

Abstract Objectives This study aimed to characterize susceptibility profiles of both currently approved and investigational β-lactam agents against difficult-to-treat resistant Pseudomonas aeruginosa (DTR P. aeruginosa) clinical isolates, including carbapenemase-producing strains. Patients and methods A total of 502 consecutive DTR P. aeruginosa clinical isolates were collected from unique hospitalized patients between 2022 and 2024 across three tertiary care centres. Isolates originated from bloodstream, respiratory, urinary and intra-abdominal infections. Minimum inhibitory concentrations (MICs) for seven β-lactam agents were determined using triplicate reference broth microdilution. Susceptibility interpretations were based on CLSI criteria or, where applicable, investigational breakpoints. Whole-genome sequencing was performed to detect β-lactamase genes. Results Among the 502 isolates, cefepime-zidebactam exhibited the highest in vitro activity (100% susceptibility), followed by cefiderocol-xeruborbactam (98%), cefiderocol (95%), ceftolozane-tazobactam (90%), cefepime-taniborbactam (86%), ceftazidime-avibactam (85%), and imipenem-relebactam (41%). Notably, only 35% of isolates were susceptible to all seven agents. Carbapenemase genes were identified in 4% of isolates, including blaGES (n = 13), blaVIM (n = 7), blaKPC (n = 1) and blaIMP (n = 1). Among carbapenemase-producing isolates, cefepime-zidebactam retained activity against all isolates (100%), exceeding that of other approved β-lactam/β-lactamase inhibitor combinations (0%–24% susceptible), cefiderocol (76%), cefiderocol-xeruborbactam (86%) and cefepime-taniborbactam (81%). Conclusions While currently approved β-lactam agents retained activity against many DTR P. aeruginosa isolates, their performance was markedly compromised in the presence of carbapenemases, with the notable exceptions of cefepime-zidebactam and cefiderocol. Investigational β-lactam/β-lactamase inhibitor combinations exhibited consistently robust activity across resistance phenotypes, including carbapenemase-producing isolates, representing promising options for DTR P. aeruginosa infections.

Abstract We report an eightfold increase in sulbactam-durlobactam MICs in a carbapenem-resistant Acinetobacter baumannii cerebrospinal fluid isolate recovered after 14 days of sulbactam-durlobactam plus meropenem therapy. Treatment-emergent mutations in the AdeFGH efflux system were identified, accompanied by a 166-fold increase in adeG expression. Efflux pump inhibition restored sulbactam-durlobactam susceptibility.

ABSTRACT Metallo-beta-lactamase (MBL)-producing Enterobacterales are a global public health concern. Infections caused by these pathogens are often hard to treat, with few effective agents currently available. While aztreonam-avibactam only recently gained U.S. and European regulatory approval, laboratories have implemented laboratory-developed tests (LDTs) using existing aztreonam and ceftazidime-avibactam reagents. We sought to compare the performance of four phenotypic methods, up to three reagent manufacturers, and two media manufacturers. One hundred two MBL-producing Enterobacterales isolates were evaluated at two sites (Centers for Disease Control and Prevention [CDC] and Johns Hopkins University [JHU] School of Medicine). The performance of four methods was evaluated: broth disk elution (BDE), disk stacking (DS), gradient diffusion strip stacking (SS), and a strip-to-disk proximity (strip-disk) method, which were compared to reference broth microdilution (rBMD) tested in parallel. Respective category agreement for CDC and JHU was 92.2% and 96.1% for BDE, 51%–76.5% and 31%–49% for DS, 71.6%–86.3% and 83.3%–86.3% for SS, and 82.4%–87.3% and 80%–89.2% for the strip-disk method. The SS essential agreement ranged from 46.1% to 62.7% for JHU and 54.9% to 92.2% for CDC. Interlaboratory reproducibility was 93.1% for rBMD, 92.2% for BDE, 47.1%–53.1% for DS, 76.5%–80% for SS, and 87.3%–88.9% for strip-disk. BDE was the only method that exceeded acceptable performance of >90% accuracy and reproducibility. DS had the lowest and most variable performance between sites and among the different disk manufacturers used. LDTs for combination testing can be highly variable, and further work is needed to improve standardization and harmonization to achieve accurate and reliable results for clinical purposes. IMPORTANCE Carbapenem-resistant Enterobacterales are classified as an urgent public health threat in the Centers for Disease Control and Prevention’s 2019 Antibiotic Resistance Threats Report. Among the most concerning resistance mechanisms are carbapenemase enzymes encoded on mobile genetic elements, which can spread rapidly between bacteria. Of the various carbapenemases, metallo-beta-lactamases are particularly challenging, as they confer resistance to nearly all beta-lactams, and current treatment options remain limited. While aztreonam-avibactam has been U.S. Food and Drug Administration (FDA)-approved for treating certain infections with limited or no alternative treatment options, no FDA-cleared susceptibility test is yet available. In this context, laboratory-developed methods, such as combination testing of aztreonam and ceftazidime-avibactam, play a critical role in guiding therapy. Our study evaluates several of the more common laboratory-developed tests compared to reference broth microdilution and found that broth disk elution was the most accurate; in contrast, other methods had variable performance, some demonstrating limited reliability and being prone to overcalling resistance.

Abstract Background Colonization with extended-spectrum β-lactamase-producing Enterobacterales (ESBL-E) is increasingly recognized in critically ill patients and impacts infection prevention practices and empiric antibiotic selection. However, contemporary United States estimates of ESBL-E prevalence at the time of ICU admission are limited.Table 1.ICU-Specific Prevalence of ESBL Colonization at Admission Confirmed by Whole Genome SequencingFigure 1.Distribution of ESBL Gene Variants Among ESBL-Producing Enterobacterales Isolates by Bacterial Species (n = 173) Methods We conducted a prospective molecular epidemiology study of ESBL-E colonization among adult and pediatric patients admitted to nine ICUs at The Johns Hopkins Hospital between March and August 2023. Routine rectal surveillance swabs (Copan Eswab) collected at ICU admission were plated on selective chromogenic media (HardyCHROM™ ESBL) to identify third-generation cephalosporin-resistant Enterobacterales (3GCRE). Species identification was performed using MALDI-TOF MS, and ceftriaxone resistance was confirmed by broth microdilution. Whole-genome sequencing (WGS) was performed on the first isolate of each species per patient to confirm ESBL gene carriage and assess molecular features. Results Among 3,278 ICU admissions, 328 (10%) had 3GCRE isolated on admission swabs. Of these isolates, 173 (5% of all admissions) were confirmed by WGS to harbor at least one ESBL gene—representing approximately 53% of all 3GCRE isolates. ESBL-E colonization was most prevalent in the medical (10%) and cardiac (8%) ICUs. The most common ESBL-E species were Escherichia coli (72%) and Klebsiella pneumoniae (19%). Dominant E. coli sequence types were ST131 (36%), ST1193 (7%), and ST38 (6%). Nearly all ESBL genes belonged to the blaCTX-M family (95%), with blaCTX-M-15 as the predominant allele (63%). Other ESBL genes included blaCTX-M-27 (17%), blaCTX-M-14 (5%), blaCTX-M-55 (5%), and blaSHV-12 (3%). Conclusion In this large, prospective cohort, 5% of ICU patients were colonized with ESBL-E at admission, most commonly E. coli and K. pneumoniae harboring blaCTX-M genes. Phenotypic detection of 3GCRE overestimated ESBL-E prevalence by nearly 90%, highlighting the need for molecular confirmation to accurately characterize resistance. These findings suggest ESBL-E colonization remains uncommon in the U.S., even at a major referral center, supporting non-carbapenem empiric therapy in low-risk patients and reinforcing the need for targeted surveillance. Disclosures All Authors: No reported disclosures

Abstract Objectives Evaluation of differences in the intestinal microbiome and resistome among high-risk patients (i.e. intensive care, oncology, transplant recipients) who are and are not colonized with carbapenem-resistant Enterobacterales (CRE). Methods One hundred and twelve rectal swabs were obtained from 85 patients with known CRE colonization status and cohorted. Long-read metagenomic next-generation sequencing (mNGS) was performed on rectal swabs. Microbiome and resistome analysis were performed by assessing α-diversity, β-diversity, relative abundance assessment and linear discriminant analysis effect size (LEfSe), comparing patients colonized (CRE positive) and not colonized (CRE negative) with CRE. Longitudinal analysis of sequential swabs collected over multiple hospital encounters on a subset of patients was performed at the patient level. Results The microbiomes of cohorts were similar when comparing α- and β-diversity measures and relative abundance. LEfSe analysis identified Gram-negative pathobionts enriched among CRE-positive samples and Gram-positive taxa enriched among CRE-negative samples. α-Diversity of the resistome differed at class, gene and allele levels. Relative abundance and LEfSe analysis demonstrated enrichment of genes conferring β-lactam resistance among CRE-positive patients; LEfSe also demonstrated enrichment of antimicrobial resistance genes to multiple antimicrobial classes. At the patient level, fluctuations in the microbiome and resistome among longitudinally collected swabs were associated with antibiotic exposure. Conclusions Differences between the microbiomes of CRE-positive- and CRE-negative-colonized patients at the cohort level were relatively muted, whereas statistically significant differences were observed among their resistomes. In patients followed longitudinally, shifts in microbiome and resistome composition were dramatic in between encounters and antibiotic exposures.

Abstract Background Pseudomonas aeruginosa has the ability to exhibit resistance to a broad range of antibiotics, highlighting the importance of identifying alternative or adjunctive treatment options, such as phages. Patients and methods We report the case of a 25-year-old male who experienced an accidental electrocution resulting in exposed calvarium in the left parieto-temporal region, complicated by a difficult-to-treat P. aeruginosa (DTR-P. aeruginosa) infection. Cefiderocol was the sole antibiotic with consistent activity against six bacterial isolates obtained from the infected region over a 38 day period. Results WGS analysis identified a blaGES-1 gene as well as the MDR efflux pumps MexD and MexX in all six of the patient’s ST235 DTR-P. aeruginosa isolates, when compared with the reference genome P. aeruginosa PA01 and a P. aeruginosa ST235 isolate from an unrelated patient. After debridement of infected scalp and bone, the patient received approximately 6 weeks of cefiderocol in conjunction with IV phage Pa14NPøPASA16. Some improvement was observed after the initiation of cefiderocol; however, sustained local site improvement and haemodynamic stability were not achieved until phage was administered. No medication-related toxicities were observed. The patient remains infection free more than 12 months after completion of therapy. Conclusions This report adds to the growing literature that phage therapy may be a safe and effective approach to augment antibiotic therapy for patients infected with drug-resistant pathogens. Furthermore, it highlights the importance of the GES β-lactamase family in contributing to inactivation of a broad range of β-lactam antibiotics in P. aeruginosa, including ceftolozane/tazobactam, ceftazidime/avibactam and imipenem/relebactam.

Plasmid-mediated colistin resistance (PMCR) is a global public health concern, given its ease of transmissibility. The purpose of this study was to evaluate two methods for the detection of PMCR from bacterial colonies: (i) the NG-Test MCR-1 lateral flow immunoassay (LFA; NG Biotech, Guipry, France) and (ii) the EDTA-colistin broth disk elution (EDTA-CBDE) screening test method. These methods were evaluated using a cohort of contemporary, clinical Gram-negative bacillus isolates from 3 U.S. academic medical centers (126 isolates of the Enterobacterales , 50 Pseudomonas aeruginosa isolates, and 50 Acinetobacter species isolates; 1 isolate was mcr positive) and 12 mcr -positive CDC-FDA Antibiotic Resistance (AR) Isolate Bank isolates for which reference broth microdilution colistin susceptibility results were available.

Susceptibility testing of the polymyxins (colistin and polymyxin B) is challenging for clinical laboratories. The Clinical and Laboratory Standards Institute (CLSI) Antimicrobial Susceptibility Testing Subcommittee evaluated two methods to enable accurate testing of these agents. These methods were a colistin broth disk elution (CBDE) and a colistin agar test (CAT), the latter of which was evaluated using two inoculum volumes, 1 μl (CAT-1) and 10 μl (CAT-10).